| 1 BackgroundPresently, due to the increase of patients with trauma and bone defect induced by high energy injury, bone tissue engineering has emerged as the time require. Base on engineering and life sciences, the purpose of tissue engineering is to repair bone defect by engineering bone tissue, which was synthesized by natural or artificial biodegradable scaffold and cells, including mesenchymal stem cells and osteoblasts. In varieties of biological materials, synthetic bone substitute materials, porous calcium phosphate biomaterials play representative role. Porous calcium phosphate biomaterials special preparation, pore structure and surface characteristics of materials etc are osteoinductive properties. At present, porous calcium phosphate biomaterials for bone induction process and mechanism by inside and outside the country of a large number of scholars to explore more than a certain theoretical basis, but the induction mechanism still exist many unsolved mysteries. This topic we try to on mesenchymal stem cells in the induction of porous calcium phosphate biomaterials for bone were studied, further understanding of porous calcium phosphate biomaterials with bone induction mechanism, so that it can be better used in clinical patients with bone defect repair. 2 ObjectiveUsing the cell membrane fluorescent dye pkh-26 and PKH-67, MSCs were labeled and traced in vivo to study the osteoinductive function of porous calcium phosphate biomaterials on MSCs. This study can help us to understandthe mechanism,of porous calcium phosphate biomaterials with bone induction and promote the clinical applicationof the porous calcium phosphate biomaterials on high energy injury or inflammation of bone defect. 3 Materials and Methods 1) Isolation, culture and identification of rabbit bone marrow mesenchymal stem cells.BMSCs were isolated from rabbit by density gradient centrifugation and subculturedand identified, to ensure that the final experiment used cells for the division anddifferentiation potential of cells with multidirectional. 2) Fluorescent labeling BMSCs3 generation of BMSCs were digested to single cell suspension by trypsin and were labeled by pkh-26 and PKH-67 according to sigma instruction manual, and experiments were proceeded as follows: flow cytometry detect BMSCs labeled rate; II labeled cells were inoculated in culture dish and placed the coverslip culture plate and fluorescence microscopy to observe the labeling of cell suspension liquid and 24 h after cell marker effect; 3 Detection of labeled BMSCs biological characters;(4) and bone Jin Shizhi spirit composite graft in rabbit muscle, 3, 7, 12, 15 weeks were not decalcification resin package embedded sections, fluorescence microscope was used to observe and identify pkh-26 and PKH-67 body tracer fluorescence effect and tracing time. 3) Animal SurgeryThe experimental rabbits were randomly divided into three groups, 12 in each group, group A for the blood vessels, group B for the local injection group and the C group as the control group. After the operation, record the order and mark. After the operation, the animal were breed in center of Lanzhou General Hospital of the Western Theater of war and given adequate food and drinking water, feeding cage to keep dry, postoperative observation. 4) BMP-2 gene expression measurementAfter the experiment animals were killed, and the samples were taken from the surrounding tissue of 1cm, and the fresh samples of the soft tissues were removed. The expression of BMP-2 gene was analyzed by RT-PCR. 5) Histological observationExperimental animals were sacrificed after harvested 4% paraformaldehyde, embedded in paraffin, Masson staining and alkaline Phospatase immunohistochemical and collagen I immune group were bone viability and new bone formation in the experimental group. 6) Statistical analysisUsing the statistical software SPSS 19.0 for, single factor variance analysis(ANOVA), data((?)ąs) standard difference said, between groups was used for pairwise comparison LSD test. P<0.05 was significantly different, with statistical significance. 4 The results of the study 1) BMSCs can stable spread to the third generation, BMSCs CD 90 was positive expression detected by flow cytometry, and the positive rate is 95.24%. CD 45 was negative expression. PKH-26 and PKH-67 fluorescent labeling real-time fluorescence microscope observation found that BMSCs into a circle, fluorescent evenly distributed on the surface of the cell membrane; 24 BMSCs after H fluorescent stable expression, the cells were spindle shaped. MTT detection and osteogenesis and fat inducing test showed that the fluorescent labeling did not affect its biological characteristics. PKH-26 and PKH-67 positive rates were 93.65% and 91.37%. 2) Bone marrow mesenchymal stem cell labeling in vivo tracing.After 3, 7, 12, 15 weeks,PKH-26 and PKH-67 fluorescence can be stably expressed in fluorescence microscopy. fluorescence light showed a wide distribution of red or green labeled tissue sections. Strength and stability of PKH-26 fluorescent protein at week 15, the fluorescence intensity of PKH-67 was slightly weakened. 3) The postoperative experiments with rabbit with air embolism and sacrificed, hard tissue slicing fluorescence microscope observation showed that, group A in 3, 7 and 12 weeks fluorescence distribution, stable expression of strength, group B in 3 weeks of distribution, 7 to 12 weeks tend to be evenly distributed control group C no fluorescence; BMP-2 gene table up to the amount determination of experimental group A, group B and control group(Group C) in different time periods of BMP-2 gene expression were statistically significant(P < 0.05), between a group and B group in different time difference has statistical significance(P < 0.05). 4) Histological results of Masson staining showed that A and B in the experimental group and C group control group from the 7 week all trabecular bone formation but new bone and collagen fiber tissue volume at each time node from the A group to the C group. The results of immunohistochemistry showed the expression of I ALP and COL to comply with the above principles. 5 The conclusion of the study 1) The experimental separation and cultivation of BMSCs, by morphological observation, flow cytometry identification, cells for BMSCs, composite standard. 2) PKH-26 and PKH-67 as a fluorescent marker of BMSCs, does not affect the morphology, proliferation and differentiation of BMSCs. 3) Porous calcium phosphate biomaterials induced micro environment of mesenchymal stem cells to artificial bone material attached, and proliferation and differentiation into bone cells, and the formation of new bone, around the capillaries and humoral mesenchymal stem cells are the source of bone forming cells and capillaries derived mesenchymal stem cells play a major role. 4) The results of this study can lay the foundation for the next step in vivo experiment. |
PDF Full Text Request